This core will be responsible for the construction of retroviral vectors and for the culturing of and genetic modification of rat and monkey primary skin fibroblasts to be used for intracerebral grafting as described in projects 1 and 3. The molecular biology part of this core will also provide assistance (e.g. in situ hybridization, polymerase chain reaction) to the projects as needed. Adeno-, adeno-associated and lentivirus (e.g. human immunodeficiency virus) based vectors expressing transgenes of interest will be made for in vivo gene transfer in the brain. The efficiency, safety, and level of transgene expression will be ascertained. In addition to the service components, the core will carry out research projects to explore vector systems for achieving long-term expression of transgenes at a physiologically significant level both in vitro and in vivo. Most gene therapy approaches have focused on using retroviral vectors to transduce foreign genes into cells. However, there is down-regulation of transgene expression from retroviral vectors in quiescent serum starved cells in vitro and in grafted cells in vivo where similar environmental condition may exist. To up-regulate transgene expression, a number of vectors containing elements in their enhances that can be regulated by steroids will be examined. A tetracycline regulatable vector system which allows the transgene expression to be shut off or turn on in a reversible manner by the effector will also be explored. A reverse system where the transgene expression can be up-regulated with tetracycline will be constructed and examined. To determine the best conditions needed to achieve optimum regulatable expression of the foreign gene products in modified fibroblasts, cells grown under various culture conditions and in the presence of different concentrations of the ligands will be examined. The research done in this core will enable us to find the best vector systems for ex vivo and in vivo gene transfer in the brain.